Phytochemical Content of Centaurea Polypodiifolia Boiss. Var. Polypodiifolia
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Int. J. Sec. Metabolite, Vol. 4: 3 (2017) pp. 452-458 Special Issue 2: Research Article ISSN: 2148-6905 online Journal homepage: http://www.ijate.net/index.php/ijsm Phytochemical Content of Centaurea polypodiifolia boiss. var. polypodiifolia Ferda ESER *1, Ayse SAHIN YAGLIOGLU2, Ebru AKTAS3, Adem ONAL3, Ibrahim DEMIRTAS2 1 Department of Private Security and Protection, Suluova Vocational Schools, Amasya University, Amasya, Turkey 2 Department of Chemistry, Faculty of Science, Cankiri Karatekin University, 18100 Cankiri, Turkey 3 Department of Chemistry, Faculty of Science & Art, Gaziosmanpasa University, 60240 Tokat, Turkey Received: 04 May 2017 – Revised: 18 September 2017 - Accepted: 03 November 2017 Abstract: Various solvents including hexane, CHCl3, EtOAc, and MeOH were used to extract the aerial parts of C. polypodiifolia Boiss. var. polypodiifolia. Hexane extract was analyzed by GC-MS and nonacosane and octacosane were determined as the main constituents. HPLC-TOF/MS analyses were carried out on CHCl3, EtOAc, and MeOH extracts. The main constituents of CHCl3 extract were detected as 4-hydroxybenzoic acid, vanilic acid, and gentisic acid. Gentisic acid, fumaric acid, chlorogenic acid, and vanilic acid were determined as the main compounds of EtOAc extract. Finally, main constituents of MeOH extract were found as fumaric acid, chlorogenic acid, quercetin-3-β-D-glucoside, gentisic acid and diosmin. Keywords: Centaurea polypodiifolia, extract, GC-MS, HPLC-TOF/MS 1. INTRODUCTION The genus Centaurea which belongs to Asteraceae family, comprises nearly 500 species around the World [1]. Centaurea species are generally used in folk medicine due to their pharmacological properties such as fever treatment, diabetes, hemoroid, and peptid ulcer [2]. Biological activity studies of Centaurea genus revealed that they have exhibited antioxidant, antimicrobial and antipyretic activities [3]. Phytochemical studies of Centaurea species showed that the main compound groups of different Centaurea species were sesquiterpenes [4-8], lignin compounds [9-11], flavonoids [12-14], and their glycosides. Secondary metabolites especially phenolic compounds are responsible from the biological [15-18] activities. Phenolic and flavonoid compounds, as important phytochemicals, are present in vegetables, fruits and cereal grains. Phytochemicals such as phenolic and flavonoid compounds commonly found in plants have multiple biological effects and play an important role in the defense against cardiovascular disease, aging and cancer [19]. In recent years, the studies related with plant-derived phytochemicals and their biological activities have gained interest that they give natural solutions for health-care [20]. *Corresponding Author E-mail: [email protected] ISSN: 2148-6905 online /© 2017 DOI: 10.21448/ijsm.376888 452 International Journal of Secondary Metabolite, Vol. 4:3 (2017) pp. 452-458 Therefore, determination of the phytochemical content of the plants is an important step for the treatment and prevention of some diseases. The objective of the current study is to determine the phytochemical content of different extracts of Centaurea polypodiifolia boiss. var. polypodiifolia. So, it facilitates the future investigations related with biological activity. 2. MATERIAL and METHODS 2.1 General Experimental Procedures GC-MS analysis was performed with a Perkin Elmer Clarus 500 Series GC system. Phenolic constituents of the extracts were determined using an Agilent Technologies 6210 Time-of-Flight LC-MS. All solvents used in HPLC analysis were of HPLC grade and purchased from Merck. The extraction solvents were distilled before the extraction processes. 2.2. Plant Material C. polypodiifolia Boiss. (CPP) was collected from Karacayir/Sivas (39 51 29 N, 36 58 48 E, 1568 m). Identification of the plant material was performed by Prof. Dr. Necati CELİK from Cumhuriyet University and Prof. Dr. Neriman OZHATAY from Istanbul University. A vouchermen deposited the plant material in the herbarium of Cumhuriyet University and Istanbul University, respectively (CUFH 8934; ISTE 85428). 2.3. Extraction Procedure Flowers of CPP (100 g) were extracted with hexane (1 L x 3) for 24 h. After the process, it was filtered and evaporated. The residue was re-extracted with the same procedure for the following solvents CHCl3, EtOAc, and MeOH, respectively. 2.4. Methylation Procedure The fatty acid composition of hexane extract was determined by using methylation of the lipid extracts to form fatty acid methyl esters (FAME) for gas chromatography (GC) analysis. For this purpose, 40 mg of extracted oil was dissolved in a tube which contained n-heptane. Then, 2 M KOH (2 mL) was added to the tube, the mixture was shaken vigorously and allowed to reach formation of phases. The upper layer, which contained the FAME was transferred to a vial and diluted with n-hexane for GC analyses [21]. 2.5. Gas Chromatography (GC) Analysis FAME analyses were carried out with a Perkin Elmer Clarus 500 Series GC system equipped with flame ionization detector (FID) and a TR-FAME apolar capillary column (30 m x 0.25 mm and 0.25 m ID). The split ratio was 50:1 port. Helium was used as the carrier gas at a flow rate of 0.5 mL/min. The temperatures of the injector and detector were set to 250 and 260 °C, respectively. An initial column oven temperature of 100 °C was raised to 220 °C at a rate of 2 °C/min. Identification of the FAME peaks was performed by comparing the retention times of each peak with those of authentic standards (Supelco 37 Comp. Fatty acid Mix, 18919) and their mass spectral 2.6. HPLC-TOF/MS Analysis Phenolic constituents of the plant extracts were determined in an Agilent 1260 Infinity HPLC system coupled with an Agilent 6210 TOF-MS detector and an VYDAC C18 column (25 mm × 300 mm 10 μm). Water (with 2.5 % formic acid) and acetonitrile were used for the mobile phases, A and B, respectively. The column temperature was adjusted to 35°C where the flow rate was 0.8 mL min-1. The injection volume was 200 μL and the elution program was as 453 Eser, Sahin, Yaglioglu, Aktas, Onal & Demirtas follows: 0–1 min, 10% B; 1–12 min, 40% B; 12–14 min, 90% B; 14–17 min, 90% B; 17–18 min, 10% B; 18–25min, 10% B. 3. RESULTS 3.1. Fatty Acid Content GC-MS analysis results of the hexane extract of CPP was given in Table 1. Main constituent of the hexane extract of CPP was assigned as nonacosane (43.53%). Table 1. Fatty acid contents of the hexane extract of CPP No RT Compound Area (%) Saturated fatty acids 1 19.771 Lauric acid 0.51 2 22.804 Myristic acid 1.21 3 27.456 Palmitic acid 3.76 4 32.491 Stearic acid 0.85 5 34.763 Arachidic acid 1.05 6 37.2 Behenic acid 0.86 7 38.688 Tricosylic acid 0.12 8 40.468 Lignoceric acid 0.50 9 45.383 Cerotic acid 0.33 10 65.035 Melissic acid 0.19 Mono unsaturated fatty acid 11 32.188 Oleic acid 2.85 Poly unsaturated fatty acid 12 32.096 Linoleic acid 2.28 Other compounds 13 19.903 Myristicin 1.41 14 25.138 3,7,11,15-Tetramethyl-2-hexadecen-1-ol 0.15 15 25.304 Hexahydrofarnesyl acetone 0.13 16 32.342 Phytol 0.40 17 33.315 Docosane 0.27 18 34.448 Tricosane 3.76 19 34.683 11-Eicosenoic acid, methyl ester 0.43 20 35.552 Tetracosane 0.70 21 35.924 Methyl 14-methyl-eicosanoate 0.11 22 36.8 Pentacosane 3.11 23 37.126 Methyl 11-docosenoate 0.18 24 38.167 Hexacosane 0.47 25 39.964 Octacosane 16.42 26 44.788 Nonacosane 43.53 27 45.583 8-Androsten-3-ol, 17-(2-methylallyl)-4,4,14- 0.22 trimethyl- 28 47.488 Triacontane 1.76 29 51.608 Tetratriacontane 9.29 30 56.197 Hexatriacontane 0.30 31 59.934 Stigmasterol 0.49 32 62.406 Hentriacontane 0.67 33 63.504 γ-Sitosterol 0.92 34 65.501 β-Amyrin 0.15 35 68.694 α-Amyrin 0.48 Total 99.86 454 International Journal of Secondary Metabolite, Vol. 4:3 (2017) pp. 452-458 3.2. Phenolic Compounds of the CPP Extracts The study was conducted to investigate the phenolic compounds of CHCl3, EtOAc, and MeOH extracts of CPP. The phenolic compound profile of CPP extracts were given in Table 2. The results of the HPLC-TOF/MS analysis revealed that the main constituents of CHCl3 extract were 4-hydroxybenzoic acid (0.168 g kg-1), followed by vanilic acid (0.133 g kg-1), and gentisic acid (0.112 g kg-1). Main phenolic compounds of EtOAc extract were gentisic acid (1.220 g kg- 1), fumaric acid (0.990 g kg-1), chlorogenic acid (0.866 g kg-1), and vanilic acid (0.571 g kg-1). HPLC-TOF/MS results of MeOH extract showed that the main constituents of the extracts were fumaric acid (137.010 g kg-1), chlorogenic acid g kg-1), quercetin-3-β-D glucoside (4. 407 g kg- 1), gentisic acid (2.567 g kg-1), and diosmin (1.954 g kg-1), respectively. It is observed that, while the main phenolic constituents of MeOH extract are determined as fumaric acid, chlorogenic acid, quercetin-3-β-D glucoside, gentisic acid, and diosmin; EtOAc extract doesn’t contain these phenolics except gentisic acid (Table 2). Table 2. Phenolic compounds of CPP extracts determined by HPLC-TOF/MS CPP Compound CHCl3 (g/kg) EtOAc (g/kg) MeOH(g/kg) Chlorogenic acid 0.866 16.305 4-hydroxybenzaldehyde 0.020 Vanilic acid 0.133 0.571 0.797 Gentisic acid 0.112 1.220 2.567 4-hydroxybenzoic acid 0.168 0.404 1.533 Kafeic acid 0.333 0.756 Rutin 0.021 Protocatechuic acid 0.184 0.951 Naringenin 0.011 0.046 0.411 Salisilic acid 0.005 Wogonin 0.007 Fumaric acid 0.990 137.010 Quercetin-3-β-D glucoside 0.287 4.407 Sinapic acid 0.081 0.143 Diosmin 0.364 1.954 Morin 0.129 0.634 The standard compounds used for HPLC-TOF/MS analysis include the following phenolics besides the phenolics in Table 2: hesperidin, apigenin-7-glucoside, rosmarinic acid, protocatechuic acid ethyl ester, caftaric acid, quercetin, p-coumaric acid, kaempferol, ferulic acid, chicoric acid, ellagic acid, resveratrol, biochanin, eupatorin, cinnamic acid, syringic acid, apigenin, scutellarin, and neohesperidin.